Neurofilaments (NF) are characteristic structures in normal neurons that become abnormally prominent and distributed in certain neuroicgical disorders and toxin-induced neuropathies. While clearly important in the development and maintenance of neuronal shape and intracellular organization their precise molecular function and the regulation thereof is not clear. The objectives of this project are to elucidate the key features of NF metabolism and how they are regulated in intact neurons. A further goal is to relate specific aspects of NF metabolism to events in the differentiation maturation and pathological alterations of the neuronal cytoskeleton. Biochemical and cytoiogical approaches will be used to examine the regulation of NF synthesis. Postransational modification and intracellular distribution during in vivo and in vitro differentiation as well as in mature neurons maintained in culture. Radioactive amino acid incorporation will be used to analyze the synthesis of NF proteins as compared with that of other neuronal cytoskeletal proteins tubulin and actin NF phosphorylation will be analyzed particularly the posttransiational phosphorylation of the avian middle Mr NF polypeptide, NF-M. This NF component is abundantly expressed in cultured neurons and is extensively phosphorylated over a prolonged time in culture and in vivo. It is proposed that the several sites become phosphorylated after translation in a specific sequence and that the different sites vary in the rate at which the subsequently exchange. This will be examined by establishing the kinetics of 32p uptake into the intact NF polypeptides as well as into individual proteolytic fragments. The effects of agents that perturb NF metabolism will be investigated, including Li+, which inhibits NF phosphorylation, tubulin metabolism and neurite growth. Analysis of this action of Li+, will assess in understanding basic aspects of reuroral cytoskeletal interactions as well as the clinically relevant mechanism of Li+, toxicity. Distribution of NF and other elements of the cytoskeleton will be examined by immunohistochemistry. Using antibodies specific to individual NF proteins at the light and EM level. The results of these experiments will provide insight into the functional significance of NF in normal neurons and in pathological conditions.